Universal car-t cell targeting il13ralpha2, preparation method therefor and application thereof

Abstract

The present application relates to a modified immune effector cell, wherein the functions of a T cell antigen receptor (TCR) and major histocompatibility complexes (MHCI, MHCII) in the modified immune effector cell are inhibited in a T cell, and the modified immune effector cell comprises a chimeric antigen receptor (CAR) targeting IL13Rα2. The modified immune effector cell of the present application knocks out TCR and HLA-A genes expressed by the cell while recognizing surface antigens of tumor cells, so that multiple effects of improving the anti-tumor effect of CAR-T cells, prolonging the survival time of the cells, and reducing the immune rejection response caused by allogeneic cell therapy are achieved.

1 . An immune effector cell, wherein the immune effector cell comprises:
 a modified immune effector cell,   wherein an expression or activity of a TRAC gene and an HLA-A gene in the modified immune effector cell is down-regulated as compared to a corresponding unmodified immune effector cell,   wherein an expression or activity of a CIITA or a B2M gene in the modified immune effector cell is not down-regulated as compared to the corresponding unmodified immune effector cell,   wherein the immune effector cell comprises a chimeric antigen receptor (CAR) targeting IL13Rα2,   wherein the chimeric antigen receptor (CAR) targeting IL13Rα2 comprises:   a targeting moiety comprising an antibody heavy chain variable region (VH),   wherein the VH comprises:
 a heavy chain complementarity-determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 1: 
 a heavy chain complementarity-determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 2; and 
 a heavy chain complementarity-determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 3, 
   wherein the chimeric antigen receptor (CAR) targeting IL13Rα2 comprises a targeting moiety comprising a light chain variable region (VL), and   wherein the VL comprises:
 a light chain complementarity-determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 19; 
 a light chain complementarity-determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 20; and 
 a light chain complementarity-determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 21. 
   
     
     
         2 - 7 . (canceled) 
     
     
         8 . The immune effector cell according to  claim 1 , wherein the VH comprises:
 a heavy chain framework region 1 (HFR1);   a heavy chain framework region 2 (HFR2);   a heavy chain framework region 3 (HFR3); and   a heavy chain framework region 4 (HFR4),   wherein the HFR1 comprises an amino acid sequence set forth in SEQ ID NO: 4, or   the HFR2 comprises an amino acid sequence set forth in SEQ ID NO: 5, or   the HFR3 comprises an amino acid sequence set forth in SEQ ID NO: 6, or   the HFR4 comprises an amino acid sequence set forth in SEQ ID NO: 7.   
     
     
         9 - 15 . (canceled) 
     
     
         16 . The immune effector cell according to  claim 1 , wherein the VH comprises:
 an HFR1;   an HFR2;   an HFR3; and   an HFR4, and   wherein the HFR1, HFR2, HFR3, and HFR4 are selected from the group consisting of   i) the HFR1 comprising an amino acid sequence set forth in SEQ ID NO: 8, the HFR2 comprising an amino acid sequence set forth in SEQ ID NO: 9, the HFR3 comprising an amino acid sequence set forth in SEQ ID NO: 10, and the HFR4 comprising an amino acid sequence set forth in SEQ ID NO: 11; and   ii) the HFR1 comprising an amino acid sequence set forth in SEQ ID NO: 12, the HFR2 comprising an amino acid sequence set forth in SEQ ID NO: 13, the HFR3 comprising an amino acid sequence set forth in SEQ ID NO: 14, and the HFR4 comprising an amino acid sequence set forth in SEQ ID NO: 15.   
     
     
         17 . The immune effector cell according to  claim 1 , wherein the VH comprises an amino acid sequence set forth in SEQ ID NO: 18. 
     
     
         18 - 25 . (canceled) 
     
     
         26 . The immune effector cell according to  claim 1 , wherein the VL comprises:
 a light chain framework region 1 (LFR1);   a light chain framework region 2 (LFR2);   a light chain framework region 3 (LFR3); and   a light chain framework region 4 (LFR4), and   wherein the LFR1 comprises an amino acid sequence set forth in SEQ ID NO: 22, or   the LFR2 comprises an amino acid sequence set forth in SEQ ID NO: 23, or   the LFR3 comprises an amino acid sequence set forth in SEQ ID NO: 24, or   the LFR4 comprises an amino acid sequence set forth in SEQ ID NO: 25.   
     
     
         27 - 33 . (canceled) 
     
     
         34 . The immune effector cell according to  claim 26 , wherein the VL comprises:
 an LFR1;   an LFR2;   an LFR3; and   an LFR4, and   wherein the LFR1, LFR2, LFR3, and LFR4 are selected from the group consisting of   i) the LFR1 comprising an amino acid sequence set forth in SEQ ID NO: 26, the LFR2 comprising an amino acid sequence set forth in SEQ ID NO: 27, the LFR3 comprising an amino acid sequence set forth in SEQ ID NO: 28, and the LFR4 comprising an amino acid sequence set forth in SEQ ID NO: 29; and   ii) the LFR1 comprising an amino acid sequence set forth in SEQ ID NO: 30, the LFR2 comprising an amino acid sequence set forth in SEQ ID NO: 31, the LFR3 comprising an amino acid sequence set forth in SEQ ID NO: 32, and an LFR4 comprising the amino acid sequence set forth in SEQ ID NO: 33.   
     
     
         35 . The immune effector cell according to  claim 1 , wherein the VL comprises an amino acid sequence set forth in SEQ ID NO: 36. 
     
     
         36 . (canceled) 
     
     
         37 . The immune effector cell according to  claim 1 , wherein the targeting moiety further comprises:
 the VH comprising an amino acid sequence set forth in SEQ ID NO: 18, and   the VL comprising an amino acid sequence set forth in SEQ ID NO: 36.   
     
     
         38 . The immune effector cell according to  claim 37 , wherein the VH and the VL are selected from the group consisting of
 i) the VH comprising an amino acid sequence set forth in SEQ ID NO: 16, and the VL comprising an amino acid sequence set forth in SEQ ID NO: 34; and   ii) the VH comprising an amino acid sequence set forth in SEQ ID NO: 17, and the VL comprising an amino acid sequence set forth in SEQ ID NO: 35.   
     
     
         39 . The immune effector cell according to  claim 1 , wherein the targeting moiety comprises a full-length antibody, a Fab, or a single-chain variable fragment (scFv). 
     
     
         40 . (canceled) 
     
     
         41 . The immune effector cell according to  claim 1 , wherein the targeting moiety comprises an amino acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 44. 
     
     
         42 . The immune effector cell according to  claim 1 , wherein the targeting moiety comprises an amino acid sequence set forth in SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 43. 
     
     
         43 - 76 . (canceled) 
     
     
         77 . The immune effector cell according to  claim 1 , wherein the chimeric antigen receptor comprises an amino acid sequence set forth in any one of SEQ ID NO: 45 and SEQ ID NO: 46. 
     
     
         78 - 93 . (canceled) 
     
     
         94 . The immune effector cell according to  claim 1 , wherein a modification comprises administering to the immune effector cell a substance selected from the group consisting of an antisense RNA, an siRNA, an shRNA, and a CRISPR/Cas9 system. 
     
     
         95 . (canceled) 
     
     
         96 . The immune effector cell according to  claim 1 , wherein a modification comprises administering to the immune effector cell an sgRNA targeting an exon portion of the TRAC gene. 
     
     
         97 . The immune effector cell according to  claim 96 , wherein the sgRNA targeting the exon portion of the TRAC gene comprises a nucleotide sequence set forth in any one of SEQ ID NO: 186 to SEQ ID NO: 200. 
     
     
         98 . The immune effector cell according to  claim 1 , wherein a modification comprises administering to the immune effector cell an sgRNA targeting an exon portion of the HLA-A gene. 
     
     
         99 . The immune effector cell according to  claim 98 , wherein the sgRNA targeting the exon portion of the HLA-A gene comprises a nucleotide sequence set forth in any one of SEQ ID NO: 201 to SEQ ID NO: 241. 
     
     
         100 - 106 . (canceled) 
     
     
         107 . A method for preparing an immune effector cell, comprising:
 modifying the immune effector cell before or after introducing a polynucleotide sequence encoding the CAR targeting IL13Rα2 according to  claim 1  or a vector comprising the polynucleotide sequence encoding the CAR targeting IL13Rα2 into the immune effector cell,   wherein the modifying comprises knocking out an exon of the TRAC gene and knocking out an exon of the HLA-A gene in the immune cell.   
     
     
         108 - 138 . (canceled) 
     
     
         139 . A pharmaceutical composition comprising the immune effector cell according to  claim 16 . 
     
     
         140 - 154 . (canceled)